JP2018501360A - Hematoxylin solution containing chloride and sulfate, and method of preparation and use - Google Patents

Abstract

The present disclosure relates to hematoxylin staining formulations, particularly formulations for use in fully automated staining systems. It has been discovered that the disclosed compositions have typical stability under storage, but have high dye quality and sufficiently fast dyeing performance. The disclosed hematoxylin staining composition includes a solvent system, hematoxylin, a chemical oxidant, and a mordant. Exemplary embodiments also have a Cl- / SO4- molar ratio between about 2.5 / 1 to about 1/4. [Selection] Figure 4 (B)

Description

  The present invention relates to compositions and methods for histochemical staining of biological samples. More specifically, the present invention relates to a hematoxylin formulation having improved stability against degradation.

  Hematoxylin has been described as the most important and most used dye in histology, histochemistry, histopathology and cytology. Several histochemical staining protocols, including hematoxylin and eosin (H & E) staining and Papanicolaou (PAP) staining, stain cytological and tissue samples depending on hematoxylin dye. In particular, hematoxylin staining of cell nuclei is used by pathologists to detect the presence of malignant and / or metastatic cells in tumor biopsy samples. Haematoxylin, natural black 1, or C.I. I. Hematoxylin, also known as 75290m, is a naturally occurring compound found, for example, in the red heartwood of the genus Acacia, of the genus Acacia. Hematoxylin is not an active ingredient that stains tissue components. Rather, hematein, an oxidation product of hematoxylin, becomes an active dye component of the hematoxylin dye solution, especially in complex formation with a mordant. Hematein-mordant complexes bind compounds containing negative charges and stain them dark blue or violet, which allows pathologists to visualize these negatively charged structures in biological samples To.

Ehrlich's hematoxylin is a widely used hematoxylin formulation because it is used to stain tissue in shades of blue, pink and red. Ehrlich's hematoxylin formulation consists of 100 mL water, 100 mL ethanol (100%), 100 mL glycerol, 10 mL glacial acetic acid, 2 g hematoxylin, and excess potassium aluminum sulfate based on solubility (alum; AlK [SO ₄ ] 2. 12H ₂ O). To make Ehrlich's hematoxylin, the ingredients are combined and allowed to age for about a month. Depending on the formulation, aging methods involving exposure to air and sunlight can provide a solution suitable for staining cells over 3 months. This method is highly variable and is typically not sufficiently reproducible with respect to the production of hematoxylin for clinical fully automated staining systems.

  Chemical oxidants can be utilized to facilitate the conversion of hematoxylin to hematein. Unfortunately, the accelerated process often produces ineffective reaction products such as oxyhematein and complex polymerization precipitates, and provides a solution that degrades faster than naturally aged dye solutions. If the exact amount of oxidant required to quantitatively oxidize hematoxylin to hematein is used, it can help avoid peroxidation to ineffective products, but if staining is not performed immediately, Partially oxidized solutions are more typically utilized. In partially oxidized solutions, the natural oxidation of hematoxylin remaining after the chemical oxidation step is either consumed during dyeing or is further naturally oxidized to ineffective products. Hematein will continue to be replaced. Nevertheless, the concentration (and amount) of hematein can change over time.

  Since hematein is an active staining component of the hematoxylin solution, changes in its concentration over time (and / or the concentration of its mordant complex) lead to inconsistent staining.

  Although hematoxylin has been known as a stain for tissues and cells for at least a century, formulations that are compatible with fully automatic staining systems have only recently been discovered. Manual staining typically involves dipping or immersing a sample mounted on a slide in a container containing a hematoxylin formulation. The first advantage of this procedure is that large sediment falls in the bottle so that it does not deposit on the slide. In manual staining procedures, changes in the hematein content of the hematoxylin solution can be compensated by changing the contact time of the biological sample with the solution based on visual inspection. For example, a sample that is clearly under-stained can simply be returned to the hematoxylin solution for a time to increase the staining intensity. In automatic staining procedures, however, extended exposure times in response to “visual” inspection and lack of staining can require expensive imaging equipment and can disrupt the processing of other samples. Despite some practical advantages of manual hematoxylin staining, patient safety concerns, reproducibility, and speed / cost have accelerated the adoption of automated hematoxylin staining. A concern for patient safety is that they may lose their adhesion to the slide during the dye bath and even lose. These extraneous samples can then be transferred to slides that can be misdiagnosed at the same time or later. The solution to this problem was to provide a disposable staining reagent as a paddle on the slide surface so that cross-contamination is not possible. The on-slide approach requires a radically different staining formulation because the delivery of reagents to the slide through a fully automated staining system uses a piping system that must maintain a clear precipitate. Furthermore, the hematoxylin solution should have an extended shelf life for commercial execution. And for high volume fully automatic staining systems, the solution may have no more than 5 minutes, typically no more than 2 minutes, to stain the sample. Thus, hematoxylin auto-staining solutions have a unique demand not met by formulations suitable for manual staining.

The present disclosure describes novel hematoxylin staining compositions and more effective tissue staining methods using such compositions. One aspect of the present disclosure is that preventing long-term staining quality while preventing precipitation in hematoxylin solutions remains a continuing challenge in the field despite many recent advances. is there. Another aspect of the present disclosure is that the use of chemical additives or any ingredient that enhances the stability of hematoxylin should be non-toxic, environmentally friendly and relatively inexpensive. Accordingly, one aspect of the present disclosure is a composition lacking toxic, environmentally harmful and relatively expensive ingredients. Instead of relying on chemical additives to slow the rate of precipitate formation, the present disclosure describes the superior results that result from the preparation of a hematoxylin solution having a defined counterion ratio. According to another aspect, sulfate as a counter ion is believed to be a presumed factor favoring precipitate formation, and inclusion of chloride as a counter ion has been discovered as a possible solution to the problem. In particular, it has been discovered that certain chloride / sulfate ratios (ie, Cl ⁻ / SO ₄ ^2— molar ratio) are particularly advantageous as dye preparations.

  Aluminum sulfate has traditionally been used in almost all hematoxylin formulations, which is believed to be because the solution prepared therefrom is superior in that it produces intense nuclear staining in a relatively short period of time. While one aspect of the present disclosure is known for aluminum sulfate and aluminum chloride based hematoxylin solutions, the examples disclosed herein are hematoxylin solutions prepared using only aluminum chloride or only aluminum sulfate. Is inferior to a composition containing the two ratios. In particular, when aluminum chloride is used as an alternative to aluminum sulfate, hematoxylin takes longer to achieve similar staining characteristics compared to aluminum sulfate formulations. Although these aluminum chloride solutions were found to be more resistant to precipitate formation, the dyeing properties were unacceptably slow. While not being limited to a particular theory, it is understood that the mechanism by which this effect is achieved is related to the greater binding affinity of chloride to aluminum compared to sulfate. It has been discovered that higher affinity (more covalent or stronger) aluminum-chloride linkages affect hemaram hue in the same way as increasing the pH of the solution for all hematoxylin solutions. It was also determined that the staining hue is affected by the chloride content. The more chlorinated ones make the dye hue from blue to red. In the sulfate-only formulation, the hematoxylin hue shifts to red as the pH is lowered (even over a narrow pH range (eg, 2.45-2.59)). In other words, the hue of the chloride-based formulation at the lower pH is similar to the hue of the conventional sulfate solution at the higher pH, and the lower pH value will favor hemaram solubility. As shown in FIG. 1 (C), functioning to interfere with the interaction between individual hemaram molecules in other cases, with the proton concentration increased at a lower working pH value than chloride allows, which adversely affects the precipitate It is understood that there is a “blocking” effect of chloride. Unfortunately, the presence of chloride (or functional staining possible at lower pH values) interferes with the formation of precipitates, but must also interfere with the desired interaction between hemaram and tissue, It is also a cause of slow dyeing speed.

  However, through careful experimentation, it was discovered that the combination of aluminum sulfate and aluminum chloride in a hematoxylin formulation dyes rapidly and strongly as a sulfate solution, but can produce a solution that is much more resistant to precipitation. . Furthermore, the examples show that the stability enhancement provided by chloride typically exceeds the benefits afforded by the use of known chemical additives (eg, host compounds and antioxidants). These additives may still be included, but their inclusion is not necessary when using the sulfate / chloride ratio disclosed herein. In yet another aspect, the present disclosure now describes non-toxic (or harmless) propylene glycol instead of ethylene glycol or ethanol (a component that some agencies classify as toxic / hazardous). It shows that it can be used in hematoxylin formulations.

Chemical structure showing a hemaram complex comprising aluminum complexed with hematein and two aqua species, such as Al ₂ (SO ₄ ) ₃ .nH ₂ O present in a hematoxylin solution used as a mordant. Chemical structure showing a hemaram oligomer showing the proposed structure for a hematoxylin precipitate in which a number of (n) hematein complexes form a chain, the inner aluminum species being complexed by two hematein species. Chemical structure showing a hemaram complex comprising aluminum complexed with hematein, one aqua species, and one chloride species, such that AlCl ₃ · 6H ₂ O is present at least partially in the hematoxylin solution used as a mordant . Photomicrograph taken at 10x magnification of serial sections of 4 micron thick tissue sections stained with freshly prepared hematoxylin solution of Example 1. Photomicrograph taken 10 times larger than a serial section of a 4 micron thick tissue section stained with a freshly prepared hematoxylin solution of Example 4. Photographed 10 times larger than a serial section of a 4 micron thick tissue section stained with the hematoxylin solution of Example 1 that underwent accelerated degradation as described in Example 9 (45 ° C., 32 days). Photomicrograph. Photographed 10 times larger than a serial section of a 4 micron thick tissue section stained with the hematoxylin solution of Example 4 that underwent accelerated degradation as described in Example 9 (45 ° C., 32 days). Photomicrograph This is the same section as shown in FIG. 2C, and is shown with a closer viewpoint (for example, digital zoom). This is the same section as shown in FIG. 2D, and is shown with a closer viewpoint (for example, digital zoom). A photograph showing the precipitation of the hematoxylin solution on the contacting surface. Shown are two bottles (modified with polyethylene terephthalate glycol) containing hematoxylin for 32 days at 5 ° C after the bottle was drained and rinsed with deionized water (DI water), the left is as described in Example 1 The bottle containing the formulation is shown, the right shows the bottle containing the formulation of Example 4. A photograph showing the precipitation of the hematoxylin solution on the contacting surface. After containing the solution at 60 ° C. for 7 days, a loop of tubing of the type (eg, perfluoroalkoxy resin (PFA)) as seen in a fully automated staining system was shown, and the loop was drained and rinsed with DI water. The left tube contained the formulation described in Example 1 and the right tube contained the formulation described in Example 4. 4 is a photomicrograph of serial sections of a 4 micron thick tissue section stained with the hematoxylin solution of Example 1 that has undergone accelerated degradation as described in Example 9 (45 ° C., 32 days). This can further explain the difference between good and poor dyeing performance described herein. 4 is a photomicrograph of a serial section of a 4 micron thick tissue section stained with the hematoxylin solution of Example 4 that has undergone accelerated degradation as described in Example 9 (45 ° C., 32 days). This can further explain the difference between good and poor dyeing performance described herein.

I. the term:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

  The singular forms “a, an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a host compound” includes one or more host compounds, such as two or more host compounds, three or more host compounds, or even four or more host compounds. The host compound of

  The term “antioxidant” refers to an atom or molecule that has a greater oxidizing ability than a second atom or molecule so that it is preferentially oxidized instead of a second atom or molecule. For example, an antioxidant may have an oxidizing capacity greater than hematein, thus helping to prevent the oxidation of hematein to oxyhematein. Furthermore, the antioxidant can also function as a reducing agent, such as a reducing agent that converts oxyhematein back to hematein.

  The term “aqueous solvent” refers to a composition that has water as a major component and is liquid at room temperature. A mixture of water having a volumetric water content of 50% or more and one or more lower alkanols or polyols is an example of an aqueous solvent. For example, a solution of ethylene glycol or propylene glycol and water is an aqueous solvent.

  The term “biological sample” refers to any sample obtained from or otherwise derived from a biological entity such as an animal, eg, a human or a livestock animal such as a dog, cat, horse or cow. Refers to the resulting sample. Examples of biological samples include cytological samples, tissue samples, and biological fluids. Specific non-limiting examples of biological samples include blood, urine, urethral gland fluid, nipple aspirate, semen, milk, sputum, mucus, pleural effusion, pelvic fluid, synovial fluid, ascites, body cavity wash, eye Brushing, skin abrasion, buccal swab, vaginal swab, pap smear, rectal swab, aspirate, needle biopsy, eg tissue section obtained by surgery or autopsy, plasma, serum, spinal fluid, lymph, sweat, tears, Includes samples obtained from saliva, tumors, organs and in vitro cells or tissue culture. Typically, the sample will be a biopsy sample that has been fixed, processed to remove water, and embedded in paraffin or another suitable waxy material to cut into tissue sections. The biological sample can be mounted on a support such as a microscope slide for processing and / or examination.

  As used herein, the term “hematoxylin composition” refers to a composition formed by directly dissolving hematein (the oxidation product of hematoxylin) in a solvent, and dissolving hematoxylin in a solvent and hematoxylin hematein. It refers generically to both compositions formed by enabling or promoting oxidation to. The disclosed composition is converted to hematein (either fully or partially) by dissolving hematoxylin in a solvent and natural or accelerated chemical oxidation by contact with air. The benefits of the stabilizing effect of the disclosed composition components can also be utilized in combination with a hematein composition prepared by directly dissolving hematein in a solvent. . Thus, in some embodiments, a “hematoxylin composition” will at least initially contain little or no hematoxylin and will consist primarily of hematein. The molar concentrations of the various components are disclosed herein. These concentrations are understood to reflect the concentration at the time of formulation and when made. The concentrations of these components will change over time according to various equilibrium biased responses. Thus, the concentration may be described when formulated or made. This approach is understood to be the clearest and most accurate way to describe the solution described herein due to the fact that it tends to age over time.

  The term “host compound” refers to an organic or inorganic molecule, complex or substance having an internal cavity or groove portion, more specifically an internal cavity portion that can accommodate at least a portion of hematein or other dye molecule or A molecule having a groove portion. Host compounds include polysaccharides such as amylose, cyclodextrins and other cyclic or helical compounds containing multiple aldose rings, such as monosaccharides (such as glucose, fructose, and galactose) and (sucrose, maltose, And compounds formed through 1,4 and 1,6 linkages of disaccharides (such as lactose). Other host compounds include cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarene, valinomycin and nigericin. In certain embodiments, the host compound can be a cyclodextrin or a cyclodextrin derivative. With respect to disclosure regarding stabilized hematoxylin solutions, US Pat. No. 8,551,731, which is incorporated by reference in its entirety, broadly discloses host compounds and their use in stabilizing hematoxylin solutions. Host compounds are effective in stabilizing the hematoxylin solution, while they tend to be expensive and a precipitate remains in the tissue. In the examples provided herein, the control composition (referred to as ANS hematoxylin) is the method disclosed in US Pat. No. 8,551,731, to represent the best known automated hematoxylin formulation currently known. It is made according to.

  Host compounds include cyclodextrin derivatives, amylose derivatives, cryptand derivatives, cryptophan derivatives, cavitand derivatives, crown ether derivatives, dendrimer derivatives, nanotube derivatives, calixarene derivatives, valinomycin derivatives, and nigericin modified with one or more substituents. Derivatives can be included. For example, host compounds include amylose derivatives and cyclodextrin derivatives in which one or more of the hydroxyl groups of the constituent aldose rings or hydrogen atoms of the hydroxyl groups are replaced by substituents. Examples of the substituent include an acyl group (acetyl group and the like), an alkyl group, an aryl group, a tosyl group, a mesyl group, an amino group (including primary, secondary, tertiary and quaternary amino groups), a halo group (- F, -Cl, -Br and -I), nitro groups, phosphorus-containing groups, (phosphoric acid and alkyl phosphate groups, etc.) sulfur-containing groups (sulfuric acid and sulfate ester groups, etc.), bridging groups (for example, cyclodextrin ring Bonding two or more hydroxyl positions, or bonding two or more host compounds.), Aldehyde groups, ketone groups, oxime groups, carboxylic acid groups and their derivatives, carbonate and carbamate groups, silicon-containing Groups, boron-containing groups, tin-containing groups, and hydroxyalkyl groups (such as hydroxyethyl and hydroxypropyl groups).

  Specific examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and δ-cyclodextrin, and the respective derivatives of these classes of cyclodextrins. Specific examples of cyclodextrin derivatives are hydroxypropylated α-cyclodextrin, hydroxypropylated β-cyclodextrin, hydroxypropylated γ-cyclodextrin, hydroxyethylated α-cyclodextrin, hydroxyethylated β-cyclodextrin, hydroxy Ethylated γ-cyclodextrin, hydroxyisopropylated α-cyclodextrin, hydroxyisopropylated β-cyclodextrin, hydroxyisopropylated γ-cyclodextrin, carboxymethylated α-cyclodextrin, carboxymethylated β-cyclodextrin, carboxymethylated γ-cyclodextrin, carboxyethylated α-cyclodextrin, carboxyethylated β-cyclodextrin, carboxyethylated γ-cycline Dextrin, octyl succinylated α-cyclodextrin, octyl succinylated β-cyclodextrin, octyl succinylated γ-cyclodextrin, acetylated α-cyclodextrin, acetylated β-cyclodextrin, acetylated γ-cyclodextrin, sulfated α -Cyclodextrin, sulfated β-cyclodextrin and sulfated γ-cyclodextrin. Other specific examples of cyclodextrin derivatives include the following β-cyclodextrin derivatives: 2,3-dimethyl-6-aminomethyl-β-cyclodextrin, 6-azido-β-cyclodextrin, 6-bromo-β- Cyclodextrin, 6A, 6B-dibromo-β-cyclodextrin, 6A, 6B-diiodo-β-cyclodextrin, 6-O-maltosyl-β-cyclodextrin, 6-iodo-β-cyclodextrin, 6-tosyl-β -Cyclodextrin, peracetyl-maltosyl-β-cyclodextrin, 6-t-butyldimethylsilyl-β-cyclodextrin, 2,3-diacetyl-6-butyldimethylsilyl-β-cyclodextrin, 2,6-dibutyl- 3-acetyl-β-cyclodextrin, 2,6-dibutyl-β-cyclo Dextrin, 2,6-t-butyl-dimethylsilyl-β-cyclodextrin and 2,6-di-O-methyl-3-allyl-β-cyclodextrin. A variety of cyclodextrins and cyclodextrin derivatives are described, for example, in CTD, Inc. (High Springs, Florida), or they can be obtained from the academic literature, for example, “Synthesis of Chemically Modified Cyclodextrins”, Craft and Bartsch, Tetrahedron, 39: 1417-1474, 1983. Can be synthesized according to

  The term “lower alkanol” refers to the formula R—OH, where R is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl. And an alkyl group having between 1 and 5 carbon atoms, such as an isopentyl group or a neopentyl group. Examples of lower alkanols include methanol, ethanol and isopropanol.

  The term “oxidant” refers to an atom or molecule that has a greater reducing ability than a second molecule, eg, a reducing ability greater than hematoxylin such that it can react with hematoxylin and oxidize to hematein. The oxidant diffuses into hematoxylin and actively binds to the naturally occurring molecular oxygen in the atmosphere that oxidizes hematoxylin and hematoxylin (typically in solution) to convert at least a portion of hematoxylin to hematein. "Oxidizing agent". Examples of useful chemical oxidants include iodate (such as sodium iodate and potassium iodate), mercuric oxide, permanganate (such as potassium permanganate), periodate (sodium periodate and Potassium peroxide, etc.), and one or more peroxides (hydrogen peroxide, etc.). In certain embodiments, the chemical oxidant comprises sodium iodate.

  The term “mordanting agent” is a complex (positive) that helps pigments (such as hematein) bind pigments (such as hematein) to specific cellular components such as nuclear DNA, myelin, elasticity and collagen fibers, striatum and mitochondria. An ionic metal species that can form an ionic complex or the like). Examples of mordants are aluminum, iron, tungsten, zirconium, bismuth, molybdenum (phosphomolybdic acid or molybdic acid), vanadium (eg in the form of alum such as aluminum sulfate, potassium aluminum sulfate, ammonium aluminum sulfate, or aluminum chloride). (Vanadate).

II. Overview Aluminum hematoxylin solutions are prone to degradation and form increasingly significant amounts of precipitate over time that can be detected in solution or on slides early in the days after preparation. FIG. 1A shows a hemaram structure derived from an aluminum sulfate mordant in an acidic solution. Hemaram is complexed with hematein and water as shown. Shown in FIG. 1B is a proposed structure of a hemaram precipitate composed of aggregates of hemaram, where the positively charged dye species is active, where n is an integer greater than 1 . On stained tissue specimen slides, the hematoxylin precipitate can exhibit a variety of forms, from discrete microcrystalline opaque spots 4-5 microns in diameter to opaque sheets greater than 100 microns in diameter (eg, a microscope It is obvious even if it is not used. The precipitate tends to collect on (ie, associate with) a negatively charged tissue component as shown in FIG. Factors understood to increase the rate of precipitation include pH values above about 2.3, elevated temperatures, freeze / thaw cycles, temperature changes, high hemaram concentrations, and extended time. Thus, maintaining a low pH, maintaining a stable temperature, and reducing the hematoxylin concentration have been used to reduce the precipitation rate. In addition, chemical stabilization techniques have recently been described (see US Pat. No. 8,551,731).

  In the solution itself, the precipitate tends to accumulate at either the air / liquid contact surface or the contact surface of the surface / container contact surface (eg on the surface of the bottle or tube). This can be seen in FIGS. 3A-B, which are photographs showing the precipitation of the hematoxylin solution on the contacting surface. FIG. 3 (A) shows two bottles (modified with polyethylene terephthalate glycol) containing hematoxylin for 32 days at 5 ° C. after draining the bottle and rinsing with DI water, the left is in Example 1. The bottles containing the described formulations are shown, the right shows the bottles containing the formulation of Example 4. FIG. 3 (B) shows a loop of tubing of the type (eg PFA) as found in a fully automated staining system after containing the solution at 60 ° C. for 7 days, the loop being drained and rinsed with DI water, The left shows a bottle containing the preparation described in Example 1, and the right shows a bottle containing the preparation of Example 4. The hematoxylin solution tends to settle on surfaces such as product packaging, forming a coating that appears copper-colored and / or having a metallic luster. Accumulation of precipitates results in significant changes in the appearance of the solutions, and acceptable staining with these solutions becomes impractical. Dissolution and prevention of precipitates is possible, for example, by acidification or chelation, but these techniques also have a negative impact on dye quality and the diagnostic utility of dyeing and therefore cannot be applied directly to dye solutions . For these reasons, natural aging cannot be used, and hematoxylin solutions are typically prepared only when needed and discarded immediately after use.

  Where longer solution lifetimes are required, such as with automated staining platforms, multiple methods have been developed to reduce the rate of precipitate formation while maintaining long-term staining properties. Most commonly, hematoxylin solutions can be prepared using sub-equivalent amounts of oxidizing agents (usually iodate). This results in less hemaram than the total possible amount initially formed, leaving unreacted dye precursor in solution. Slow “ripening” (air oxidation) of the remaining hematoxylin into hematein, the component that actually binds aluminum to form a hemaram dye, results in a lower concentration of hemaram in solution at any given time Thus delaying the formation and accumulation of precipitates. Inclusion of antioxidants in formulations (eg, hydroquinone) can also delay the aging process and provide additional formulation stability with respect to precipitate formation. Although not common, chemical additives such as beta-cyclodextrin can also be introduced to slow the rate of precipitate formation. Additives work by providing a reversible chemical pathway that kinetically competes with aggregation, thereby slowing the rate of precipitation. In the case of beta-cyclodextrin, hemaram molecules in solution form weak complexes with the additive, reducing the likelihood that hemaram molecules will complex with each other. Unfortunately, the thermodynamically desirable product (precipitate) will still be ultimately obtained in the same amount as without the additive, but at a reduced rate.

  The innovation of hematoxylin solution was recently disclosed in published patent publication No. 2014-59282 by Wadamatsu Kikuo. This published patent publication, which can be translated as “Antimicrobial Hematoxylin Solution and Expanded Antimicrobial Hematoxylin Solution and Extended Antibacterial Hematoxylin Solution”, is a hematin preparation that exhibits antibacterial / antifungal properties. Because hematoxylin compositions are not widely known to be susceptible to infection with bacteria or fungi, the disclosure of antimicrobial hematoxylin is somewhat puzzling. The solution described contains 5.0 g aluminum chloride hexahydrate; 1.5 g hematoxylin; 0.3 g sodium iodate; 30 mL ethanol; 870 mL water; 100 mL glycerol. Except for the presence of iododate and the relative amounts of ingredients, this formula is very similar to Mayer's musihematein, a hematoxylin solution formulated for manual staining using aluminum chloride as a mordant. Another common aluminum chloride containing formulation is Rosas iron hematoxylin, which also contains ammonium iron sulfate as a co-mordant.

Applicant refers to the Mayer formulation and specifically states that aluminum chloride was used because it dissolves easier (faster) than the typical aluminum mordant, aluminum potassium sulfate (potassium alum). . Although it is not explicitly disclosed why this formulation is more resistant to microorganisms / fungi, an excess amount of Al (III) is already in solution and the oxidation potential of iododate is reduced by lowering the pH of the solution. It is understood from (1) the large amount of iododate used (upper limit of the amount used in other formulations) and (2) the addition of iododate at the end of formulation. There is no suggestion that chloride is associated with microbial / fungal resistance. Rather, iododate is known as a disinfectant and high concentrations will naturally provide better bactericidal properties. With respect to the order of addition, iododate (IO ₃ ⁻ ) oxidizes hematoxylin and iodide (I ⁻ ) is formed as the final product. Some iodides are oxidized to elemental iodine (I ₂ ) by residual iodine, and this process is catalyzed by excess Al (III) in solution. For example, our experiments showed that significant amounts of iodine inevitably formed and could be clearly observed in the container when the yodate was added in the same order as reported. Since this is not preferred for our formulation, our process for producing hematoxylin defines the oxidation of hematoxylin before the aluminum is added.

Furthermore, published patent publication No. 2014-059282 discloses that dyeing is slow in chloride formulations, which is completely consistent with our results. To counter this slow staining, published patent publication No. 2014-059282 discloses mixing an aluminum chloride solution and an aluminum sulfate solution prior to the addition of hematoxylin and iodate. This solution is referred to in that patent document as “Extended Antibacterial Hematoxylin Solution” (EAHS). However, the disclosed composition still stains tissue too slowly for the fully automated staining system disclosed herein. The disclosure also does not suggest that a Cl ⁻ / SO ₄ ^2- molar ratio between about 2.5 / 1 and about 1/2 is important for stability.

III. Hematoxylin Composition In an exemplary embodiment, the hematoxylin staining composition comprises a solvent, hematoxylin, having a chloride / sulfate (Cl ⁻ / SO ₄ ²⁻ ) molar ratio between about 2.5 / 1 and about 1/4. A chemical oxidizer in an amount sufficient to convert at least a portion of the hematoxylin into a mordant. In an exemplary embodiment, the hematoxylin composition further comprises Cl ⁻ and SO ₄ ²⁻ , wherein the Cl ⁻ / SO ₄ ² molar ratio is between about 2.5 / 1 to about 1/4. , Hematoxylin, at least a portion of hematoxylin containing a chemical oxidizer, mordant sufficient to convert hematoxylin into hematein. In one embodiment, the mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ O, where M is a monovalent cation. ), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ (SO ₄ ) ₃ · 18H ₂ O, or [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O. Non-limiting examples of monovalent cations include Li ⁺ , Na ⁺ , K ⁺ , Cs ⁺ , NH ₄ ^{+ and the} like.

In one embodiment, the mordant comprises AlCl ₃ or AlCl ₃ · 6H ₂ O. In another embodiment, the mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ ), wherein M is a monovalent cation. O), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ (SO ₄ ) ₃ · 18H ₂ O, [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O, AlCl ₃ or AlCl ₃ · 6H ₂ O is included. In one embodiment, the composition has a Cl ⁻ / SO ₄ ² molar ratio of between about 2/1 to about 1/2. In another embodiment, the composition has a Cl ⁻ / SO ₄ ² molar ratio of between about 1.5 / 1 to about 1 / 1.5. In yet another embodiment, the composition has a Cl ⁻ / SO ₄ ² molar ratio of about 1/1. In one embodiment, the chemical oxidant is sodium iodate.

  In an exemplary embodiment, the hematoxylin staining composition is made to have a molar concentration of hematoxylin between about 0.01M and about 0.05M. In another embodiment, the composition is made to have a molar concentration of hematoxylin between about 0.02M and about 0.04M. In yet another embodiment, the composition is made to have a molar concentration of about 0.03 M hematoxylin. In another embodiment, the composition is made to a molar concentration of sodium iodate between about 0.001M and about 0.01M. In another embodiment, the composition is made to a molar concentration of sodium iodate between about 0.003M and about 0.008M. In one embodiment, the composition is made to have a molar concentration of about 0.005 M sodium iodate. In another embodiment, the composition is about 0.1 M aluminum. In another embodiment, the composition is made to have a molar concentration of about 0.1 M aluminum. In yet another embodiment, the composition is made to have a molar concentration of aluminum greater than about 0.1M. In another embodiment, the composition has an aluminum / hematoxylin molar ratio between about 4/1 and about 1/1. In one embodiment, the composition has an aluminum / hematoxylin molar ratio between about 3/1 and about 1.5 / 1. In another embodiment, the composition has an aluminum / hematoxylin molar ratio of about 2/1. In yet another embodiment, the composition is between about 0.01M and about 0.1M chloride. In one embodiment, the composition is between about 0.02M and about 0.08M chloride. In another embodiment, the composition is about 0.4M chloride.

  In an exemplary embodiment, the hematoxylin in the hematoxylin staining composition has a molar concentration between about 0.01M and about 0.05M. In another embodiment, the hematoxylin in the hematoxylin staining composition has a molar concentration between about 0.02M and about 0.04M. In yet another embodiment, the hematoxylin in the hematoxylin staining composition has a molar concentration of about 0.03M. In another embodiment, the composition comprises sodium iodate having a molar concentration between about 0.001M and about 0.01M. In another embodiment, the composition comprises sodium iodate having a molar concentration between about 0.003M and about 0.008M. In one embodiment, the composition comprises sodium iodate having a molar concentration of about 0.005M. In another embodiment, the composition comprises aluminum, wherein the aluminum has a molar concentration of about 0.1M. In yet another embodiment, the composition comprises aluminum, wherein the aluminum has a molar concentration greater than about 0.1M.

  In another embodiment, the composition is made to have a molar concentration of chloride between about 0.01M and about 0.1M. In one embodiment, the composition is made to have a molar concentration of chloride between about 0.02M and about 0.08M. In another embodiment, the composition is made to have a molar concentration of about 0.04M chloride.

  In another embodiment, the chloride in the hematoxylin staining composition has a molar concentration between about 0.01M and about 0.1M. In another embodiment, the chloride in the hematoxylin staining composition has a molar concentration between about 0.02M and about 0.08M. In yet another embodiment, the chloride in the hematoxylin staining composition has a molar concentration of about 0.04M.

  In one embodiment, the composition is substantially devoid of polysaccharides, cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarene, valinomycin, or nigericin. In another embodiment, the composition is substantially devoid of antioxidants. In one embodiment, the pH of the composition is between about 2 and about 2.7. In another embodiment, the pH of the composition is between about 2.2 and about 2.6.

IV. Method for making a hematoxylin composition In an exemplary embodiment, a method for making a hematoxylin formulation comprises adding hematoxylin to a solvent; adding a chemical oxidant in an amount sufficient to convert at least a portion of the hematoxylin to hematein. it; formulation ^Cl between about 2.5 / 1 to about 1/4 - with a / _SO ^{4 2-molar} ratio, comprising the addition of a mordant and counterion. In one embodiment, the mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ O, where M is a monovalent cation. ), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ (SO ₄ ) ₃ · 18H ₂ O, [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O, AlCl ₃ or AlCl containing ₃ · 6H ₂ O mixtures.

The hematoxylin composition described herein is a general method of:
1. 1. Add / dissolve hematoxylin in aqueous solution 2. Add / dissolve chemical oxidizer. 3. Add / dissolve mordant. If ^necessary, Cl - / _SO ⁴ 5 modifying the ² molar ratio. If necessary, it is produced as additional additives are added.

Thus, in certain embodiments, a method of making a hematoxylin formulation comprises adding hematoxylin to a solvent; adding a chemical oxidant in an amount sufficient to convert at least a portion of hematoxylin to hematein; mordant and counterion was ^added, Cl - comprising modifying the molar ratio between about 2.5 / 1 to about 1/4 / _SO ^{4 2-molar} ratio.

Although specific steps are disclosed in this order, they can also be done in a different order. For example, hematoxylin can be prepared in a first solution and the remaining ingredients can be made in a second solution. The final composition can then be adjusted by mixing the solutions. Further, the mordant, oxidant, and further additives can be made in a different order that has only minimal impact. An exception to this is the addition of chemical oxidants to hematoxylin. Although at least not well known, it is believed that according to some embodiments, the chemical oxidant should be added prior to the mordant. Certain other ^components, Cl - / _SO ⁴ could be used to modify the ^2-molar ratio. For example, any chloride-containing salt can be used to modify the Cl ⁻ / SO ₄ ^2- molar ratio (eg, NaCl). Alternatively, aluminum chlorosulfate, an aluminum salt with both sulfate anions and chlorine anions, may be used as a mordant and then, if appropriate, the salt is used and Cl ⁻ / SO ₄ ²⁻ The molar ratio can be further modified. Another approach to obtain the appropriate Cl ⁻ / SO ₄ ^2- molar ratio is to use the chloride salt of any ingredient in the formulation (eg, use the hydrochloride salt of the ingredient instead of the usual free base) That is).

In some cases, these methods using the compositions described herein may provide stability benefits as well. In yet another approach, the acid having a chloride counterion is added, Cl - ^/ SO ₄ ^2- mole ratio and can adjust both the pH. However, the amount of chloride that can be added using this approach will be limited by the pH limitations of the solution. In particular, the pH of the hematoxylin solution is beneficially between about 2 and about 2.7, or between about 2.2 and about 2.6.

V. Methods of Using Hematoxylin Compositions In an exemplary embodiment, a method for staining a biological sample using a fully automated staining system is described herein using a fully automated staining system. In contact with the hematoxylin composition. In one embodiment, the method further comprises contacting the sample with a counterstain. In another embodiment, the counterstain is selected from the group consisting of eosin Y, orange G, light green SF yellowish, bismarck brown, and fast green FCF. In another embodiment, the method comprising contacting the sample with a hematoxylin composition comprises a progressive hematoxylin staining protocol. In another embodiment, contacting the sample with the hematoxylin composition comprises a regressive hematoxylin staining protocol. In yet another embodiment, the biological sample is supported on a substrate. In one embodiment, the substrate comprises a microscope slide. In another embodiment, the biological sample comprises a tissue section or cytology sample. In one embodiment, the method comprises a hematoxylin and eosin (H & E) staining method. In another embodiment, the method comprises a Papanicolaou (PAP) staining method.

  All staining was performed using automated staining equipment. The instrument was used for automated cover slips and heat curing (92 ° C.). The tissue slides in all studies were Symphony SystemMulti-Block Test Slides (P / N 1707100) featuring five different tissues (liver, skin, kidney, tonsils, small intestine) mounted on a single slide. It was. Many studies used simultaneous application of both test and control formulations. The control formulation is described as ANS and is described in Example 1. Random pairs of consecutive tissue pieces were used to minimize changes in tissue morphology.

  Although various staining parameters were tested, the most commonly used procedures for each study were 2 min hematoxylin incubation (H2), 30 sec acid wash (A30), 120 sec eosin incubation (E120) and It is described as the standard “intermediate” level staining protocol H2A30E120D70 featuring 70 seconds of differentiation (D70). However, additional studies used the standard “low” and “high” level staining protocols, H1A180E30D70 and H10A30E420D70, respectively, to show that the test formulations were able to adequately meet the ANS for staining quality across all protocols. I confirmed it. At least 8 (but in most cases 16) pairs of sequentially cut tissue treated with the test and control hematoxylin solutions are then presented to the pathologist in a blinded format to perform Fisher's exact test. The number and extent of differentiation that allowed statistical significance for the changes determined using was evaluated.

  Automated staining instruments now typically filter hematoxylin before placing hematoxylin on the slide to produce a slide that contains a low non-specific signal. Another aspect of the procedure described herein is that the primary hematoxylin filter has been removed so that any precipitate in the solution appears on the slide. Thus, one aspect of the method for using the composition according to the present disclosure is to add an unfiltered composition to the sample. It has been observed that the exemplary compositions described herein, particularly in Example 4, do not exhibit precipitate on the slide, as seen in the composition of Example 1. Furthermore, the hardware of the fully automatic dyeing system likewise remained free of obvious precipitate even after the composition was stored and used for dyeing for continuous use for more than 6 weeks.

  The following examples are provided to illustrate certain specific features of the embodiments and general protocols. The scope of the invention is not limited to the features demonstrated by the following examples.

に従って作製した。 Example 1
The ANS hematoxylin formulation was based on the use of Al ₂ (SO ₄ ) ₃ .nH ₂ O as a mordant and was specifically optimized for automated H & E staining using the Ventana symphony fully automated staining system. The formulation is based on that disclosed in US Pat. No. 8,551,731. This hematoxylin formulation has the following formulations:

It produced according to.

Follow the steps below for the composition:
1. 1. Add / dissolve hematoxylin in water and ethylene glycol solution. 2. Add / dissolve sodium iodate. _3. Add / dissolve Al ₂ (SO ₄ ) ₃ · 16H ₂ O It was prepared as hydroquinone and β-cyclotextrin were added / dissolved.

  Since this composition is the best known composition, it was used as a benchmark for automated dyeability. This composition can deliver the appropriate stain on the slide in approximately 2 minutes. The composition dyes well, has the correct hue and depth, and has sufficient long-term stability, but is not ideal in all respects. In particular, the composition precipitates over time, and this effect adversely affects the dyeing equipment line. This can be mitigated through proper maintenance and cleaning, but the frequency and extent of maintenance and cleaning is considered too great. Furthermore, the precipitate may become visible on the stained slide and may even be visible. Although a precipitate is visible, it does not create a successful challenge and is therefore initially a negative aspect of this composition from an aesthetic point of view. With the addition of hydroquinone and β-cyclodextrin, the composition has a relatively high cost. And hydroquinone has been classified as a carcinogen in several jurisdictions. A typical staining using ANS hematoxylin can be seen in FIG. 2 (A). FIG. 2 (A) was stained under the following protocol.

Functional staining was assessed on symphony “5-in-1” test slides using unfiltered hematoxylin solution.

Example 2
Hematoxylin was produced having a Cl ⁻ / SO ₄ ^2- molar ratio of 1/0 (ie 100% Cl ⁻ ). The same ingredients and concentrations used in Example 1 were used except that AlCl ₃ hexahydrate was used instead of Al ₂ (SO ₄ ) ₃ · 16H ₂ O.

  Although this composition was observed to be very stable, it was also observed to have a very slow reaction rate during dyeing. Thus, for dyeing under normal operating conditions, the dyeing is very thin.

Example 3
Hematoxylin was produced having a Cl ⁻ / SO ₄ ^2- molar ratio of 1/1 (ie 50% Cl ⁻ ). In such formulations, SO ₄ ^2- causes its complete removal from ANS-Cl to result in poor staining (staining depth and hue) and unacceptable reaction rates (eg very slow automated staining). Because, included. Furthermore, the concentration of hematoxylin will be increased, Cl ^- reaction rate which is understood to be inhibited by the presence of is increased.

The formulation showed high quality dyeing and sufficient dyeing speed. After multiple experiments, it was concluded that higher concentrations of hematoxylin compared to ANS could be used to ensure that the rate of staining met the ANS benchmark. It was observed that the composition was sufficiently stable for extended periods of time without either β-cyclodextrin or hydroquinone as included in the ANS. This formulation, Cl ^- is understood to be the effect of concentration was also found to have the start of dramatically delayed precipitation.

Example 4
Although the dyeing performance and speed of ANS-Cl-50 met or exceeded the ANS benchmark performance, Example 4 was found to have even higher performance.

This formulation exceeded the performance of ANS-Cl-50 in terms of dyeing quality, dyeing kinetics, and stability, but did not contain ethylene glycol, a known dangerous substance. Non-toxic propylene glycol can be substituted for ethylene glycol, but synergistically improves dyeing quality and increases dyeing speed. Our experimental results demonstrated that a Cl ⁻ / SO ₄ ^2- molar ratio greater than 1: 1 increases stability but at the expense of slower dyeing rates. The slow reaction rate could not be justified because the stability enhancement observed for this composition was sufficient. However, such compositions would be beneficial dye compositions provided with additional time, temperature, or reaction rate. Means for increasing the dyeing reaction rate include, among other things, adjusting the pH higher, increasing the reaction temperature, or increasing the concentration of the reaction mixture.

Example 5
Various other ratios were prepared in which AlCl ₃ .6H ₂ O and Al ₂ (SO ₄ ) ₃ · 16H ₂ O were used as mordants to demonstrate the effect of the Cl ⁻ / SO ₄ ² molar ratio. . The pH was adjusted to match the ANS hue. All other components are the same as ANS (Example 1). For a 1: 4 Cl ⁻ / SO ₄ ² molar ratio (20% Cl ⁻ ), comparable staining characteristics were obtained when compared to ANS, but the reaction rate was observed to be substantially slower. Furthermore, the stability or onset of precipitation was observed to be inferior to ANS-Cl-50.

Example 6
For a 1: 2 Cl ⁻ / SO ₄ ^2- molar ratio (33% Cl ⁻ ), all components were included as described in Example 5. In addition to the change in molar ratio, the concentration of the components was increased by 25% over Example 5. It was observed that comparable staining characteristics were obtained when compared to ANS, but the reaction rate was substantially slower. Furthermore, the stability or onset of precipitation was observed to be inferior to ANS-Cl-50.

Example 7
For a 1: 2 Cl ⁻ / SO ₄ ^2- molar ratio (33% Cl ⁻ ), all components were included as described in Example 5, except that the concentration was increased by 50% over Example 5. It was. When compared with ANS, comparable staining characteristics were obtained and the reaction rate was observed to be substantially equivalent to ANS.

Example 8
Example 8 illustrates a 1: 7 Cl ⁻ / SO ₄ ² molar ratio (12.5% Cl ⁻ ). Example 8 was found to have further enhanced performance.

  The formulation exceeded the performance of NT-ANS-Cl-50 with respect to dyeing quality, dyeing kinetics, and stability and did not contain ethylene glycol. When compared with ANS, comparable staining characteristics were obtained and the reaction rate was observed to be substantially equivalent to ANS. The results obtained using gallic acid monohydrate corresponded to the results observed in the experiment (Example 4) in which gallic acid monohydrate was not used.

Example 9
A study of accelerated stability at 45 ° C. was performed only for Example 7 and Example 3, which were stained equivalent to ANS. Although Example 7 formed a precipitate as fast as ANS, it was observed that Example 3 did not form a precipitate over a 32 day study.

Example 10
The various compositions described herein were stored in sealed containers either at room temperature or 45 ° C. or for a period prior to use. Throughout the remaining examples, photomicrographs are provided that compare to various compositions and convey staining quality and degree of precipitation.

  FIG. 2 (A) is a photomicrograph taken at 10x magnification of a serial section of a 4 micron thick tissue section stained with a freshly prepared hematoxylin solution of Example 1, FIG. B) is a photomicrograph taken at 10x the serial section of a 4 micron thick tissue section stained with the freshly prepared hematoxylin solution of Example 4, FIG. , Taken 10 times larger than a serial section of a 4 micron thick tissue section stained with the hematoxylin solution of Example 1 that underwent accelerated degradation as described in Example 9 (45 ° C., 32 days). FIG. 2 (D) shows a series of 4 micron thick tissue sections stained with the hematoxylin solution of Example 4 that had undergone accelerated degradation as described in Example 9 (45 ° C., 32 days). A photomicrograph taken at 10 times the section. 2 (E) is the same section as shown in FIG. 2 (C) and is shown at a closer viewpoint (eg, digital zoom), and FIG. 2 (F) is shown in FIG. 2 (D). It is the same section as shown, shown with a closer viewpoint (eg, digital zoom).

  Functional staining with ANS hematoxylin stored at 45 ° C. revealed an increase in the amount of precipitate starting at ˜7 days. For ANS-Cl hematoxylin under the same conditions (> 6 weeks), no evidence of precipitation was detected after an extended period.

  No difference in precipitation was observed for the hematoxylin package. FIG. 3 (AB) is a photograph showing a precipitate of hematoxylin solution on the contacting surface. FIG. 3 (A) shows two bottles (modified with polyethylene terephthalate glycol) containing hematoxylin for 32 days at 5 ° C. after draining the bottle and rinsing with DI water, the left is in Example 1. The bottles containing the described formulations are shown, the right shows the bottles containing the formulation of Example 4. FIG. 3 (B) shows a tube loop of the type (eg perfluoroalkoxy resin (PFA)) as found in a fully automated staining system after containing the solution at 60 ° C. for 7 days, the loop being drained. Rinse with DI water. The left tube contained the formulation described in Example 1 and the right tube contained the formulation described in Example 4. In all cases, the ANS hematoxylin solution clearly produced more precipitate coating inside the tube loop than the chloride-based hematoxylin solution.

Referring now to FIG. 4 (A), what is shown is a 4 micron thickness stained with the hematoxylin solution of Example 1 that underwent accelerated degradation as described in Example 10 (45 ° C., 32 days). FIG. 4 is a photomicrograph of a serial section of the tissue section, referring now to FIG. 4 (B), which shows an example subjected to accelerated degradation as described in Example 10 (45 ° C., 32 days) 4 is a photomicrograph of serial sections of 4 micron thick tissue sections stained with 4 hematoxylin solutions. Referring to FIG. 4 (A), the intensity of nuclear staining seen is substantially thinner than that observed in FIG. 4 (B). Because the pathologist typically prefers a deeper nuclear staining intensity, the staining in FIG. 4 (A) is inferior to FIG. 4 (B). Furthermore, the hematoxylin precipitate that can be observed with a microscope (the upper left part of FIG. 4A) is an undesirable characteristic for observation on a sample stained with hematoxylin. Precipitates can often obscure the underlying tissue morphology, making it difficult to diagnose. In the sample shown in FIG. 4B, a hematoxylin precipitate is present, but the size of the precipitate is substantially small, obscuring the underlying tissue morphology to be less important. Furthermore, the staining intensity is still strong enough to allow proper diagnosis.
・

Claims (36)

A hematoxylin staining composition comprising a solvent, hematoxylin, a chemical oxidant and a mordant in an amount sufficient to convert at least a portion of hematoxylin into hematein, further comprising chloride (Cl ⁻ ) and sulfate (SO ₄ ²⁻ ) A hematoxylin staining composition comprising a chloride / sulfate molar ratio between about 2.5 / 1 to about 1/4. The mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ O), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ The hematoxylin according to claim 1, comprising (SO ₄ ) ₃ · 18H ₂ O or [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O, wherein M is a monovalent cation. Dyeing composition. The hematoxylin staining composition according to claim 1, wherein the mordant comprises AlCl ₃ or AlCl ₃ .6H ₂ O. The mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ O), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ (SO ₄ ) ₃ · 18H ₂ O, [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O, AlCl ₃ or AlCl ₃ · 6H ₂ O, and M is a monovalent cation The hematoxylin staining composition according to claim 1.   5. The hematoxylin staining composition according to any one of claims 1 to 4, wherein the chloride / sulfate molar ratio is between about 2/1 to about 1/2.   5. The hematoxylin staining composition according to any one of claims 1 to 4, wherein the chloride / sulfate molar ratio is between about 1.5 / 1 to about 1 / 1.5.   The hematoxylin staining composition according to any one of claims 1 to 4, wherein the chloride / sulfate molar ratio is about 1/1.   8. The hematoxylin staining composition according to any one of claims 1 to 7, wherein the hematoxylin has a molar concentration between about 0.01M and about 0.05M.   8. The hematoxylin staining composition according to any one of claims 1 to 7, wherein the hematoxylin has a molar concentration between about 0.02M and about 0.04M.   The hematoxylin staining composition according to any one of claims 1 to 7, wherein the hematoxylin has a molar concentration of about 0.03M.   The hematoxylin staining composition according to any one of claims 1 to 10, wherein the chemical oxidant is sodium iodate.   12. The hematoxylin staining composition of claim 11, wherein the sodium iodate has a molar concentration between about 0.001M and about 0.01M.   The hematoxylin staining composition of claim 11, wherein the sodium iodate has a molar concentration between about 0.003M and about 0.008M.   The hematoxylin staining composition of claim 11, wherein the sodium iodate has a molar concentration of about 0.005M.   The hematoxylin staining composition according to any one of claims 1 to 14, comprising aluminum, wherein the aluminum has a molar concentration of about 0.1M.   15. The hematoxylin staining composition according to any one of claims 1 to 14, comprising aluminum, wherein the aluminum has a molar concentration greater than about 0.1M.   17. The hematoxylin staining composition of claim 15 or 16, having an aluminum / hematoxylin molar ratio between about 4/1 and about 1/1.   17. A hematoxylin staining composition according to claim 15 or 16, having an aluminum / hematoxylin molar ratio between about 3/1 and about 1.5 / 1.   The hematoxylin staining composition according to claim 15 or 16, having an aluminum / hematoxylin molar ratio of about 2/1.   20. The hematoxylin staining composition according to any one of claims 1 to 19, wherein the chloride has a molar concentration between about 0.01M and about 0.1M.   20. The hematoxylin staining composition according to any one of claims 1 to 19, wherein the chloride has a molar concentration between about 0.02M and about 0.08M.   20. The hematoxylin staining composition according to any one of claims 1 to 19, wherein the chloride has a molar concentration of about 0.04M.   23. The hematoxylin staining composition according to any one of claims 1 to 22, substantially lacking a polysaccharide, cryptand, cryptophane, cavitand, crown ether, dendrimer, nanotube, calixarene, valinomycin, or nigericin.   24. The hematoxylin staining composition according to any one of claims 1 to 23, which is substantially devoid of antioxidants.   A method for staining a biological sample using a fully automatic staining system, wherein the hematoxylin composition according to any one of claims 1 to 24 is applied to a biological sample using a fully automatic staining system. A method comprising contacting.   26. The method of claim 25, further comprising contacting the sample with a counterstain.   27. The method of claim 26, wherein the counterstain is selected from the group consisting of eosin Y, orange G, light green SF yellowish, bismarck brown, and fast green FCF.   28. A method according to any one of claims 25 to 27, wherein contacting the sample with the hematoxylin composition comprises a progressive hematoxylin staining protocol.   28. A method according to any one of claims 25 to 27, wherein contacting the sample with the hematoxylin composition comprises a fading hematoxylin staining protocol.   28. A method according to any one of claims 25 to 27, wherein the biological sample is supported on a substrate.   32. The method of claim 30, wherein the substrate comprises a microscope slide.   32. A method according to any one of claims 25 to 31 wherein the biological sample comprises a tissue section or cytology sample.   33. A method according to any one of claims 25 to 32 comprising a hematoxylin and eosin (H & E) staining method.   33. A method according to any one of claims 25 to 32 comprising a Papanicolaou (PAP) staining method.   A method of making a hematoxylin formulation comprising adding hematoxylin to a solvent; adding a chemical oxidant in an amount sufficient to convert at least a portion of hematoxylin to hematein; adding a mordant and counterion And the formulation further comprises the step of having a chloride / sulfate molar ratio and modifying the chloride / sulfate molar ratio to a molar ratio between about 2.5 / 1 to about 1/4. The mordant is Al ₂ (SO ₄ ) ₃ , Al ₂ (SO ₄ ) ₃ · 16H ₂ O, AlM (SO ₄ ) ₂ · 12 (H ₂ O), Al ₂ (SO ₄ ) ₃ · 6H ₂ O, Al ₂ (SO ₄ ) ₃ · 18H ₂ O, [Al (H ₂ O) ₆ ] ₂ (SO ₄ ) ₃ · 5H ₂ O, AlCl ₃ or AlCl ₃ · 6H ₂ O 36. The method of claim 35, which is an ion.

Images